LHRH antagonist peptides

ABSTRACT

Novel LHRH antagonist peptides, pharmaceutical compositions thereof, and methods of use thereof, are disclosed.

This invention was made with Government support under contractN01-HD-3-3172 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

The present invention relates to LHRH antagonist peptides and usesthereof.

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) arehormones released by the pituitary gland. These hormones regulate thefunctioning of the gonads and the production and maturation of gametes.LH and FSH are generally released by the pituitary gland upon priorrelease of triggering hormone from the hypothalamus. Luteinizinghormone-releasing hormone (LHRH; also known as gonadotropin-releasinghormone or GnRH) is one of the principal hypothalamic hormones whichtriggers the release of LH. Thus, release of LHRH represents a controlpoint in the physiological regulation of gonadal function. The structureof mammalian LHRH has been determined, and has been found to be adecapeptide: ##STR1##

LH release is necessary for ovulation; thus, compounds which inhibit LHrelease by blocking the action of LHRH are useful as contraceptiveagents. LHRH antagonists are also useful for regulating secretion ofgonadotropins in male mammals, and thus can be used as malecontraceptives. In addition, LHRH antagonists can be used in thetreatment of sex-hormone dependent cancers (for example, prostatecancer), where increased levels of gonadotropins increase the rate oftumor growth.

Many modified LHRH analog peptides have been synthesized in an attemptto increase the potency of the antagonists, preferably while alsoincreasing the resistance of the antagonist to enzymatic degradation.For example, synthetic LHRH antagonist peptides which incorporatemodified or unnatural amino acids have been tested. Common substitutionsinclude, for example, substitution of 4-Cl-D-Phe for His at position 2,or substitution of D-Ala-NH₂ for Gly-NH₂ at position 10.

One problem frequently encountered in LHRH antagonist peptides is theoccurrence of histamine-releasing activity. This histamine-releasingactivity represents a serious obstacle to the clinical use of suchantagonists because histamine release results in adverse side effectssuch as edema and itching. Thus, LHRH antagonist peptides which have lowhistamine releasing activity are particularly desirable. Although theLHRH antagonist and histamine-releasing properties are not necessarilyrelated, very few prior art compounds combine low histamine-releasingactivity with high LHRH antagonist activity. Many prior art LHRHantagonist peptides also suffer from poor water-solubility, whichcomplicates formulation of the antagonist for administration.

SUMMARY

The present invention features LHRH antagonist peptides, methods ofmodulating LHRH activity, and methods of treating a subject with theantagonists of the invention. In one aspect, the invention provides anLHRH antagonist, comprising a peptide having a sidechain modified by adipolar moiety forming a modified peptide, such that the modifiedpeptide has LHRH antagonist activity.

In another aspect, the invention provides an LHRH antagonist, comprisinga peptide having a sidechain modified by a cationic moiety selected fromthe group consisting of cationic pyridinium moieties and sulfoniummoieties, with the proviso that the cationic moiety is not N-methylpyridinium, forming a modified peptide, such that the modified peptidehas LHRH antagonist activity.

In another aspect, the invention provides an LHRH antagonist, comprisinga peptide having a sidechain modified by a receptor-modifying moietyforming a modified peptide, such that the modified peptide has LHRHantagonist activity.

In still another aspect, the invention provides an LHRH antagonist,comprising a peptide having a sidechain modified by a hydrophilic N-acylmoiety forming a modified peptide, such that the modified peptide hasLHRH antagonist activity.

In yet another aspect, the invention provides an LHRH antagonist,comprising a peptide having a small polar moiety in position 6, suchthat the peptide has LHRH antagonist activity.

In still further aspects, the invention provides pharmaceuticalcompositions of the LHRH antagonist peptides.

In another aspect, the invention provides a method of inhibiting LHRHactivity in a subject, comprising administering to a subject aneffective amount of an LHRH antagonist, such that LHRH activity isinhibited.

In another aspect, the invention provides a method of inhibiting LHRHactivity in a cell, comprising contacting a cell with an LHRHantagonist, such that LHRH activity is inhibited.

In another aspect, the invention provides a method of inhibiting growthof a hormone-dependent tumor in a subject, comprising administering to asubject an effective amount of an LHRH antagonist, such that tumorgrowth is inhibited.

In another aspect, the invention provides a method of inhibitingovulation in a subject, comprising administering to a subject aneffective amount of an LHRH antagonist, such that ovulation isinhibited.

In another aspect, the invention provides a packaged formulation fortreating a subject for a disorder associated with LHRH activity,comprising an LHRH antagonist packaged with instructions for using theLHRH antagonist for treating a subject having a disorder associated withLHRH activity.

DETAILED DESCRIPTION

In order that the present invention may be more readily understood,certain terms are first defined.

As used herein, "LHRH antagonist peptide" means a peptide or peptideanalog which inhibits LHRH activity (i.e., has "LHRH antagonistactivity") in vivo or in vitro. Candidate LHRH antagonist peptides canbe assayed in the animal model described in Corbin and Beattie,Endocrine Res. Commun. 2:1 (1975) (and see infra). In this assay, theLHRH antagonistic activity of a candidate compound is assayed bymeasuring the antiovulatory activity (AOA) of the compound in rats.

The term "histamine-releasing activity", as used herein, refers to thetendency of a compound to release histamine when administered to asubject. The histamine-releasing activity of a compound can be measuredwith an in vitro assay (described in more detail, infra). Preferred LHRHantagonist peptides have high activity in the rat antiovulatory activityassay, but low histamine releasing activity. Preferred LHRH antagonistpeptides have an ED₅₀ in the histamine release assay of at least 3μg/ml, more preferably at least 5 μg/ml, and still more preferably atleast 10 μg/ml.

The term "alkyl", as used herein, refers to a straight or branched chainhydrocarbon group having from about 1 to about 10 carbon atoms. The term"lower alkyl" refers to an alkyl group having from about 1 to about 6carbon atoms. Exemplary lower alkyl groups include methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,and n-hexyl. An alkyl group may be unsubstituted, or may be substitutedat one or more positions, with, e.g., halogens, alkyls, cycloalkyls,alkenyls, alkynyls, aryls, heterocycles, hydroxyls, aminos, nitros,thiols, amines, imines, amides, phosphonates, phosphines, carbonyls,carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers, ketones,aldehydes, esters, --CF₃, --CN, or the like. Preferred alkyls are loweralkyls.

The term "cycloalkyl" refers to a cyclic saturated hydrocarbon grouphaving from 3 to 8 carbon atoms. Exemplary cycloalkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl.Cycloalkyl groups may be unsubstituted or substituted at one or morering positions as described for alkyls. Thus, a cycloalkyl may besubstituted with, e.g., halogens, alkyls, cycloalkyls, alkenyls,alkynyls, aryls, heterocycles, hydroxyls, aminos, nitros, thiols amines,imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls,ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters,--CF₃, --CN, or the like.

The terms "alkenyl" and "alkynyl", as used herein, refer to unsaturatedgroups analogous in length and possible substitution to the alkylsdescribed above, but which contain at least one carbon--carbon double ortriple bond respectively.

The term "aryl" as used herein includes 4-, 5-, 6- and 7-memberedsingle-ring aromatic groups which may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine,pyridazine and pyrinidine, and the like. Those aryl groups havingheteroatoms in the ring structure may also be referred to as "arylheterocycle" or "heteroaromatic". The aromatic ring can be substitutedat one or more ring positions with such substituents as described above,as for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls,heterocycles, hydroxyls, aminos, nitros, thiols, amines, imines, amides,phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers,thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, --CF₃,--CN, or the like. An aromatic ring may also be substituted with anotheraromatic ring, as in, for example, a biphenyl. Aryl groups also includefused or polycyclic aromatic systems.

The terms "heterocycle" or "heterocyclic group" refer to 4- to10-membered ring structures, more preferably 5 to 7 membered rings,which ring structures include one to four heteroatoms. Heterocyclicgroups include pyrrolidine, oxolane, thiolane, imidazole, oxazole,piperidine, piperazine, morpholine. The heterocyclic ring can besubstituted at one or more positions with such substituents as describedabove, as for example, halogens, alkyls, cycloalkyls, alkenyls,alkynyls, aryls, other heterocycles, hydroxyl, amino, nitro, thiol,amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls,silyls, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes,esters, --CF₃, --CN, or the like. Heterocycles may also be bridged orfused to other cyclic groups as described below.

The terms "polycycle" or "polycyclic group" refer to two or more cyclicrings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocycles) in which two or more carbons are common to two adjoiningrings, e.g., the rings are "fused rings". Rings that are joined throughnon-adjacent atoms are termed "bridged" rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls,hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates,phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls,selenoethers, ketones, aldehydes, esters, --CF₃, --CN, or the like.

The term "heteroatom" as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,sulfur, phosphorus and selenium.

The term "arylalkyl", as used herein, refers to an aryl group appendedto an alkyl group, including, but not limited to, benzyl,naphthylmethyl, pyridyl methyl, and the like.

The term "ylid" is known in the art and refers to a moiety in which apositively charged atom (especially from Groups V and VI of the periodictable) is bonded to a carbon atom which bears an unshared pair ofelectrons. Thus, an ylid has the resonance forms: ##STR2## in which W isa heteroatom such as S, or P, and R₁ and R₂ are, independently, H,alkyl, cycloalkyls, alkenyl, alkynyl, aryl, alkoxy, thioalkoxy, and thelike. The heteroatom is substituted with an appropriate number ofsubstituents (i.e., two for N and S, and three for P); the substituentsare independently alkyl, cycloalkyl, aryl, and the like. Nitrogen ylidesdo not have a significant contribution from the non-charge-separatedresonance form (on the right, above).

The term "dipolar moiety", as used herein, refers to a covalently bondedmoiety having both positive and negative charges (e.g., a zwitterionicmoiety). Exemplary dipolar groups include ylids (e.g., of S, N, or P),tertiary amine oxides, nitrones, pyridine-N-oxides, nitrile oxides,quaternary amino acids (e.g., 2-(N,N,N-trialkylammonium) acetate), aminoacids, sulfonium arene oxides (as described in, for example, U.S. Pat.No. 4,111,914), betaines (e.g., trigonellin), and the like. In certainpreferred embodiments, the dipolar moiety is a pyridine-N-oxide. Inother preferred embodiments, the dipolar moiety is a zwitterionicpyridinium moiety.

As used herein, a "cationic moiety" is a moiety in which at least oneatom bears a positive charge, and the moiety has a net positive charge.Thus, for example, an N-alkyl (or N-alkenyl, -alkynyl, or -aryl,collectively referred to herein as "N-substituted pyridinium")pyridinium moiety can be a cationic moiety (and is referred to herein asa "cationic pyridinium moiety"), but a pyridine-N-oxide is not, unlessit has a net positive charge. As described above, a pyridine-N-oxide canbe a dipolar moiety. Other exemplary cationic moieties includequaternary amines, sulfonium salts, phosphonium salts, and the like. Incertain preferred embodiments, the cationic moiety is a sulfoniummoiety.

A sulfonium moiety has the following structure: ##STR3## in which R₃, R₄and R₅ are each, independently, alkyl, cycloalkyl, alkenyl, alkynyl,aryl, and the like.

In other preferred embodiments, the cationic moiety is a cationicpyridinium moiety. A cationic pyridinium moiety has the followingstructure: ##STR4## wherein R₁₂ is alkyl or aryl, and R₁₃ -R₁₇ are each,independently, hydrogen, halogens, alkyls, cycloalkyls, alkenyls,alkynyls, aryls, other heterocycles, hydroxyl, amino, nitro, thiol,amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls,silyls, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes,esters, --CF₃, --CN, or the like. Preferred cationic pyridinium moietiesinclude Pal(iPr) and Pal(Bzl). N-methyl pyridinium moieties are notpreferred.

Although the above examples describe pyridine (or pyridinium) moieties,it will be apparent to the skilled artisan that other N-heteroaromaticmoieties (that is, a moiety in which at least one nitrogen is present inan aromatic ring) may be substituted for the pyridine (or pyridinium)moieties described herein. Exemplary N-heteroaromatics include thiazole,triazole, tetrazole, pyrazole, pyrazine, pyridazine and pyrimidine, andthe like. Thus, N-substituted pyrazines, pyridazines, and the like, arecontemplated for use in the present invention.

As used herein, "tertiary amine" includes trialkyl amines, triarylamines, and amines which have both alkyl and aryl substituents.

As used herein, "zwitterionic pyridinium moiety" refers to a moietyhaving the form: ##STR5## in which R₆ comprises an alkyl, cycloalkyl,alkenyl, alkynyl, or aryl moiety, and R₇ -R₁₁ are each, independently,hydrogen, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls,other heterocycles, hydroxyl, amino, nitro, thiol, amines, imines,amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers,thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, --CF₃,--CN, or the like, with the proviso that at least one of R₆ -R₁₁ issubstituted with an anionic moiety. An "anionic moiety", as used herein,is a moiety which has a net negative charge. The anionic moiety ischosen to be compatible with other moieties, and to form a stablecompound. Illustrative anionic moieties include carboxylates,phosphates, phosphonates, sulfates, sulfonates, and the like. In certainpreferred embodiments, the anionic moiety is a carboxylate. In otherpreferred embodiments, the anionic moiety is a sulfonate. In a preferredembodiment, R₆ comprises: ##STR6##

A pyridine N-oxide is a moiety which has the form: ##STR7## where R₁₃-R₁₇ have the meanings defined above.

The term "hydrophilic N-acyl moiety", as used herein, refers to a moietywhich comprises a nitrogen atom acylated so as to form a hydrophilicmoiety. Thus, a hydrophilic N-acyl moiety can have the form: ##STR8##where R₄₁ and R₄₂, are each, independently, H, alkyl, cycloalkyl, aryland the like; and R₄₃ is alkyl, cycloalkyl, alkenyl, alkynyl, aryl, andthe like; and R₄₁ -R₄₃ are selected to form a hydrophilic moiety. Inpreferred embodiments, R₄₁ and R₄₂ are not both H.

Relative hydrophilicity can be determined by any of several methodsknown in the art (Hansch, ed., "Comprehensive Medicinal Chemistry", Vol.4, Pergamon Press, Oxford, 1990), and can be used to guide the choice ofpotential hydrophilic moieties for use in the invention. The partitioncoefficient, P, between 1-octanol and water has been used as a referencefor measuring the hydrophilicity of a compound. Hydrophilicity can beexpressed as log P, the logarithm of the partition coefficient (Hanschet al., Nature 194:178 (1962); Fujita et al., J. Am. Chem. Soc. 86:5175(1964)). Standard tables of hydrophilicity for many molecules, andlipophilicity (hydrophobicity) substituent constants (denoted π) formany functional groups, have been compiled (see, e.g., Hansch and Leo,"Substituent Constants for Correlation Analysis in Chemistry andBiology," Wiley, New York, N.Y., (1979)). The hydrophilicity of a vastrange of candidate hydrophilicity moieties can be quite accuratelypredicted with the aid of these tables. For example, the measured log P(octanol/water) of naphthalene is 3.45. The substituent constant p for--OH is -0.67. Therefore, the predicted log P for β-naphthol is3.45+(-0.67)=2.78. This value is in good agreement with the measured logP for β-naphthol, which is 2.84. In certain preferred embodiments, thehydrophilic N-acyl moiety has a value of log P between -1 and +2, morepreferably between -0.5 and +1.5. Examples of residues incorporatingpreferred hydrophilic acyl moieties are D-Lys(Imdac), D-Lys(Ppic) andD-Lys(Dodac). Even more preferred residues incorporating hydrophilicacyl moieties include Lys(pGlu), Lys(Otac), and Lys(Onic).

The term "small polar moiety" refers to a moiety which has small stericbulk and is relatively polar. Polarity is measured as hydrophilicity bythe P scale described above. In certain preferred embodiments, the smallpolar moieties have a log P between -1 and +2. In particularly preferredembodiments, the small polar moiety modifies residue 6. In preferredembodiments, the steric bulk of the small polar moiety is less than thesteric bulk of Trp. Examples of residues incorporating preferred smallpolar moieties are D- or L-Cit, D- or L-Asn, D- or L-Gln, and D- orL-Thr. In preferred embodiments, the small polar moiety is not Glu or acarboxylic ester of Glu.

The term "leaving group" is known in the art and, as used herein, refersto a functionality which upon heterolytic bond cleavage departs with anelectron pair. In general, good leaving groups are those moieties whichare expelled from the substrate as weak bases, whether charged oruncharged. For example, sulfates, sulfonates, sulfides, chloride,bromide, iodide, phosphates, phosphinates, and the like are good leavinggroups. In other words, when, for example, a C--S bond of a sulfoniummoiety is cleaved, a sulfide departs (with an electron pair).

The term "receptor-modifying moiety", as used herein, refers to a moietywhich can modify, covalently or non-covalently, a receptor for an LHRHantagonist peptide. For example, it has recently been shown (C. A.Flanagan et al., (1994) J. Biol. Chem. 269:22636) that Glu301 of themouse LHRH receptor (which corresponds to Asp301 in the human LHRHreceptor) interacts with Arg⁸ in LHRH agonists. Thus, acarboxylate-modifying reagent (such as an alkylating agent) can modifyGlu301 (Asp301) and thus modify the mouse (or human) LHRH receptor. Areceptor-modifying moiety may act to bond the LHRH antagonist peptide tothe receptor (e.g., esterifying the antagonist to the receptor bydisplacement of a leaving group), or it may modify the receptor withoutbonding the LHRH antagonist peptide to the receptor (e.g., bymethylation with a methylsulfonium moiety). Other residues of an LHRHreceptor can also be modified, and moieties which can modify suchresidues are also receptor-modifying moieties. Exemplaryreceptor-modifying reagents include alkyl and benzyl halides (e.g.,methyl iodide or benzyl bromide), α-haloketones, α-haloesters andα-haloamides (collectively referred to as "α-halocarbonyls"), sulfoniumsalts, sulfates, alkyl or aryl sulfonates, and other reagents whichcomprise a good leaving group as described above. Otherreceptor-modifying reagents are described in, for example, A. J. Barrettand G. Salvesen, eds. (1986) "Proteinase Inhibitors", ResearchMonographs in Cell and Tissue Physiology, Vol. 12, Elsevier Press,Amsterdam.

Although in certain embodiments an LHRH antagonist of the inventioncontains a receptor-modifying moiety, the invention is not intended tobe limited to those antagonists that actually modify a receptor residue.An LHRH antagonist comprising a receptor-modifying moiety but which doesnot actually modify the receptor may nonetheless be an effective LHRHantagonist. However, for those antagonists that do modify a receptorresidue, one advantage is that such moieties can be designed toselectively modify only the targeted receptor, thereby reducingnon-specific reactions and decreasing the probability of toxic sideeffects.

The term "a peptide having a sidechain modified by" a moiety, as usedherein, refers to a peptide (or peptide mimetic, see below) in which atleast one residue has a sidechain comprising that moiety. Thus, forexample, a "peptide having a sidechain modified by a dipolar moiety"means a peptide in which at least one side chain comprises a dipolarmoiety.

The LHRH antagonist peptides of the present invention also includepeptide analogs and peptide mimetics. The terms "peptide analog","peptide derivative" and "peptide mimetic" as used herein are intendedto include molecules which mimic the chemical structure of a peptide andretain the functional properties of the peptide. A "residue" refers toan amino acid or amino acid mimetic incorporated in the peptide compoundby an amide bond or amide bond mimetic. Approaches to designing peptideanalogs are known in the art. For example, see Farmer, P. S. in DrugDesign (E. J. Ariens, ed.) Academic Press, New York, 1980, vol. 10, pp.119-143; Ball. J. B. and Alewood, P. F. (1990) J. Mol. Recognition 3:55;Morgan, B. A. and Gainor, J. A. (1989) Ann. Rep. Med. Chem. 24:243; andFreidinger, R. M. (1989) Trends Pharmacol. Sci. 10:270.

An "amino acid mimetic" refers to a moiety, other than a naturallyoccurring amino acid, that conformationally and functionally serves as asubstitute for a particular amino acid in a peptide compound withoutadversely interfering to a significant extent with the function of thepeptide (e.g., interaction of the peptide with an LHRH receptor). Insome circumstances, substitution with an amino acid mimetic may actuallyenhance properties of the peptide (e.g., interaction of the peptide withan LHRH receptor). Examples of amino acid mimetics include D-aminoacids. LHRH antagonist peptides substituted with one or more D-aminoacids may be made using well known peptide synthesis procedures. Theeffect of amino acid substitutions with D-amino acids or otheramino-acid mimetics can be tested using assays, e.g., the AOA andhistamine-release assays as described below. Other methods ofdetermining the effect of substitution with an amino acid mimetic willbe apparent to the skilled artisan.

The peptide analogs or mimetics of the invention include isosteres. Theterm "isostere" as used herein refers to a sequence of two or moreresidues that can be substituted for a second sequence because thesteric conformation of the first sequence fits a binding site specificfor the second sequence. The term specifically includes peptideback-bone modifications (i.e., amide bond mimetics) well known to thoseskilled in the art. Such modifications include modifications of theamide nitrogen, the α-carbon, amide carbonyl, complete replacement ofthe amide bond, extensions, deletions or backbone crosslinks. Severalpeptide backbone modifications are known, including ψ CH₂ S!, ψ CH₂ NH!,ψ C(S)NH₂ !, ψ NHCO!, ψ C(O)CH₂ !, and ψ (E) or (Z) CH═CH!. In thenomenclature used above, ψ indicates the absence of an amide bond. Thestructure that replaces the amide group is specified within thebrackets. Other examples of isosteres include peptides substituted withone or more benzodiazepine molecules (see e.g., James, G. L. et al.(1993) Science 260:1937-1942).

Other possible modifications include an N-alkyl (or aryl) substitution(ψ CONR!), backbone crosslinking to construct lactams and other cyclicstructures, or retro-inverso amino acid incorporation (ψ NHCO!). By"inverso" is meant replacing L-amino acids of a sequence with D-aminoacids, and by "retro-inverso" or "enantio-retro" is meant reversing thesequence of the amino acids ("retro") and replacing the L-amino acidswith D-amino acids. For example, if the parent peptide is Thr-Ala-Tyr,the retro modified form is Tyr-Ala-Thr, the inverso form is thr-ala-tyr,and the retro-inverso form is tyr-ala-thr (lower case letters refer toD-amino acids). Compared to the parent peptide, a retro-inverso peptidehas a reversed backbone while retaining substantially the originalspatial conformation of the side chains, resulting in a retro-inversoisomer with a topology that closely resembles the parent peptide and isable to bind the selected LHRH receptor. See Goodman et al."Perspectives in Peptide Chemistry" pp. 283-294 (1981). See also U.S.Pat. No. 4,522,752 by Sisto for further description of "retro-inverso"peptides.

In addition to amino acid-substituted LHRH antagonist peptides, theinvention also encompasses LHRH antagonist peptide compounds havingother modifications. For example, the amino-terminus or carboxy-terminusof the peptide can be modified. The term "amino-derivative group" isintended to include amino-terminal modifications of the peptidecompounds of the invention. Examples of N-terminal modifications includealkyl, cycloalkyl, aryl, arylalkyl, and acyl groups. A preferredN-terminal modification is acetylation. The N-terminal residue may belinked to a variety of moieties other than amino acids such aspolyethylene glycols (such as tetraethylene glycol carboxylic acidmonomethyl ether), pyroglutamic acid, succinoyl, methoxy succinoyl,benzoyl, phenylacetyl, 2-, 3-, or 4-pyridylalkanoyl, aroyl, alkanoyl(including acetyl and cycloalkanoyl e.g., cyclohexylpropanoyl),arylakanoyl, arylaminocarbonyl, alkylaminocarbonyl,cycloalkylaminocarbonyl, alkyloxycarbonyl (carbamate caps), andcycloalkoxycarbonyl, among others.

The term "carboxy-derivative group" is intended to includecarboxy-terminal modifications of the peptide compounds of theinvention. Examples of modifications of the C-terminus includemodification of the carbonyl carbon of the C-terminal residue to form acarboxyterminal amide or alcohol (i.e., a reduced form). In general, theamide nitrogen, covalently bound to the carbonyl carbon on theC-terminal residue, will have two substitution groups, each of which canbe hydrogen, alkyl or an alkylaryl group (substituted or unsubstituted).Preferably the C-terminal is an amido group, such as --CONH₂, --CONHCH₃,--CONHCH₂ C₆ H₅ or --CON(CH₃)₂, most preferably --CONH₂, but may also be2-, 3-, or 4-pyridylmethyl, 2-, 3-, or 4-pyridylethyl, carboxylic acid,ethers, carbonyl esters, alkyl, arylalkyl, aryl, cyclohexylamide,piperidineamide, other mono or disubstituted amides, ureas, orcarbamates. Other moieties that can be linked to the C-terminal residueinclude piperidine-4-carboxylic acid or amide and cis- ortrans-4-amino-cyclohexanecarboxylic acid or amide.

The modified forms of LHRH antagonist peptides of the invention,including L- or D-amino acid substitutions, covalent modification of endtermini or side chains, and peptide analogs and mimetics can be selectedfor desired alterations of the physical or chemical properties of thepeptide, for example, increased stability, solubility, bioavailability,increased or decreased immunogenicity, etc. The peptides of theinvention can be targeted to particular organs (e.g. the brain) bymethods known in the art, for example, the dihydropyridine-pyridiniumcarrier of Bodor (see, e.g., U.S. Pat. No. 4,540,564). In an exemplaryembodiment, when a side-chain modified by a pyridinium moiety isdesired, the corresponding N-alkylated dihydropyridine sidechain isincorporated into the peptide. When the petide is administered to asubject, the N-alkylated dihydropyridine sidechain is oxidized in vivoto the desired pyridinium moiety.

Preferred LHRH antagonist peptides of the present invention range inlength from about 8 to about 12 residues, more preferably from 9 to 11residues, and most preferably are 10 residues in length.

The LHRH antagonist peptides of the present invention can be prepared byany suitable method for peptide synthesis, including solution-phase andsolid-phase chemical synthesis. Preferably, the peptides are synthesizedon a solid support. Methods for chemically synthesizing peptides arewell known in the art (see, e.g., Bodansky, M. Principles of PeptideSynthesis, Springer Verlag, Berlin (1993) and Grant, G. A (ed.).Synthetic Peptides: A User's Guide, W.H. Freeman and Company, New York(1992). Automated peptide synthesizers are commercially available.

The use of combinatorial libraries to identify ligands is now wellestablished (see, e.g., M. A. Gallop et al., (1994) J. Med. Chem.37:1233; and E. M. Gordon et al., (1994) J. Med. Chem. 37:1385; andreferences cited therein). Therefore, LHRH antagonist peptides can beidentified by chemical (e.g., solution or solid-phase) synthesis ofcombinatorial libraries (e.g., of peptides or peptoids) and screening ofthe resulting libraries according to known techniques. Thus, manypotential ligands can be synthesized and screened in a short period oftime, and the most active ligands selected for further testing or use.

Standard abbreviations and conventions are used throughout thisdisclosure when describing the peptides of the invention. Peptides arewritten with the N-terminus on the left, the carboxyl terminus on theright. Amino acids are of the L-form unless stated otherwise, e.g.,D-Lys means the D-form of lysine. Ac-Xaa means the N-terminal residueXaa is N-acetylated; C-terminal amides are denoted Xaa-NH₂. In Table 1,only residues which differ from native mammalian LHRH are noted; thus,the notation Met(S⁺ Me)⁸ -LHRH.TFA means a peptide which differs fromnative mammalian LHRH only in the substitution of Met(S⁺ Me) for thenative Arg at position 8 (TFA indicates the trifluoroacetate salt).Lys(iPr) denotes N-ε-2-propyl-lysinyl; other alkylating and acylatingmoieties are similar indicated. Thus, for example, Met(S+CH₂ C₆ H₅)denotes S-benzyl methionine. Certain other non-standard residues andmoieties are abbreviated as follows:

    ______________________________________                                        Abbreviation  Residue or moiety                                               ______________________________________                                        pGlu          pyro-glutamyl                                                   Nal           3-(2-naphthyl)alaninyl                                          Ada           3-(1-adamantanyl)alaninyl                                       4-Cl--Phe     (4'-chlorophenyl)alaninyl                                       Qal           3-(2'-quinolinyl)alaninyl                                       Pal           3-(3'-pyridyl)alaninyl                                          Pal(N--O)     3-(3'-pyridine-N-oxide)alaninyl                                 Pal(iPr)      3-N-(2-propyl)-3'-pyridinium)alaninyl                           Pal(Bzi)      3-N-(benzyl)-3'-pyridinium)alaninyl                             Pal(CH.sub.2 COO.sup.-)                                                                     3-(3'-pyridinium-N-(2-acetate))alaninyl                         Lys(iPr)      N-ε-2-propyl-lysinyl                                    Imdac         2-oxo-4-imidazolinyl                                            Otac          2-oxo-4-thiazolinyl                                             Ppic          3-(piperidin-1-yl)-propanoyl                                    Dodac         2,5-dioxo-4-imidazolinyl                                        Met(S.sup.+ Me)                                                                             S-methyl methioninyl                                            PEG           polyethylene glycol                                             Cit           citrullinyl                                                     Glu(Taurine)  5-(2-sulfoethylamido)glutamyl                                   Pyz           1,4-pyrazinecarbonyl                                            Pip           pipecolyl                                                       CNa           (2-cyano)acetyl                                                 Dea           diethylamide                                                    Onic          3-nicotinyl-N-oxide                                             Glc           gluconate                                                       Orotic        orotate                                                         Orn           ornithine                                                       Dap           2,4-diaminopropionyl                                            ______________________________________                                    

I. LHRH Antagonist Peptides of the Invention

In one aspect, the invention pertains to LHRH antagonist peptides.

In one embodiment, the invention provides a peptide comprising astructure:

    A-B-C-D-E-F-G-H-I-J                                        (SEQ ID NO: 2)

wherein

A is pyro-Glu, Ac-D-Nal, Ac-D-Qal, Ac-Sar, or Ac-D-Pal

B is His or 4-Cl-D-Phe

C is Trp, D-Pal, D-Nal, L-Nal, D-Pal(N-O), or D-Trp

D is Ser

E is N-Me-Ala, Tyr, N-Me-Tyr, Ser, Lys(iPr), 4-Cl-Phe, His, Asn, Met,Ala, Arg or Ile;

F is ##STR9## wherein R and X are, independently, H or alkyl; and

Y comprises a dipolar moiety;

G is Leu or Trp;

H is Lys(iPr), Gln, Met, or Arg

I is Pro; and

J is Gly-NH₂ or D-Ala-NH₂ ;

or a pharmaceutically acceptable salt thereof. In preferred embodiments,Y is selected from the group consisting of ylids, tertiary amine oxides,nitrile oxides, pyridine-N-oxides, and pyridinium zwitterions. Inparticularly preferred embodiments, Y is an ylid, a pyridine-N-oxide ora pyridinium zwitterion. In a preferred embodiment, the peptidecomprises a structure:

    Ac-D-Nal-4-Cl-Phe-D-Pal-Ser-Tyr-D-Pal(N-O)-Leu-Lys(iPr)-Pro-D-Ala-NH.sub.2.

In a preferred embodiment, the peptide comprises a structure:

    Ac-D-Nal-4-Cl-D-Phe-D-Pal-Ser-Tyr-D-Pal(CH.sub.2 COO-)-Leu-Lys(iPr)-Pro-D-Ala-NH.sub.2 ;

or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a peptide comprising astructure:

    A-B-C-D-E-F-G-H-I-J                                        (SEQ ID NO: 3)

wherein

A is pyro-Glu, Ac-D-Nal, Ac-D-Qal, Ac-Sar, or Ac-D-Pal

B is His or 4-Cl-D-Phe

C is Trp, D-Pal, D-Nal, L-Nal, D-Pal(N-O), or D-Trp

D is Ser

E is N-Me-Ala, Tyr, N-Me-Tyr, Ser, Lys(iPr), 4-Cl-Phe, His, Asn, Met,Ala, Arg or Ile;

F is D-Arg, D-Lys(iPr), D-Pal(iPr), D-Cit or Q, wherein Q has astructure ##STR10## wherein R and X are, independently, H or alkyl; and

Z comprises a cationic moiety selected from the group consisting ofcationic pyridinium moieties and sulfonium moieties, with the provisothat the cationic moiety is not N-methyl pyridinium;

G is Leu or Trp;

H is Lys(iPr), Gln, Met, Arg or Q;

I is Pro; and

J is Gly-NH₂ or D-Ala-NH₂ ;

with the proviso that at least one of F and H is Q;

or a pharmaceutically acceptable salt thereof.

In preferred embodiments, F is Q and Z is a cationic pyridinium moiety.In preferred embodiments, Z is an N-benzyl pyridinium moiety. In otherpreferred embodiments, F is Q and Z is a sulfonium moiety. In yet otherpreferred embodiments, H is Q and Z is a sulfonium moiety.

In a particularly preferred embodiment, the peptide comprises astructure

    Ac-Sar-4-Cl-D-Phe-D-Nal-Ser-Tyr-D-Pal(Bzl)-Leu-Lys(iPr)-Pro-D-Ala-NH.sub.2 ;

or a pharmaceutically acceptable salt thereof.

In a particularly preferred embodiment, the peptide comprises astructure:

    Ac-D-Nal-4-Cl-D-Phe-D-Trp-Ser-Tyr-D-Met(S.sup.+ Me)-Leu-Arg-Pro-D-Ala-NH.sub.2 ;

or a pharmaceutically acceptable salt thereof.

In a particularly preferred embodiment, the peptide comprises astructure:

    Ac-D-Nal-4-Cl-D-Phe-D-Pal-Ser-Tyr-D-Arg-Leu-Met(S.sup.+ Me)-Pro-D-Ala-NH.sub.2 ;

or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a peptide comprising astructure:

    A-B-C-D-E-F-G-H-I-J                                        (SEQ ID NO: 4)

wherein

A is p-Glu, Ac-D-Nal, Ac-D-Qal, Ac-Sar, or Ac-D-Pal

B is His or 4-Cl-D-Phe

C is Trp, D-Pal, D-Nal, L-Nal, D-Pal(N-O), or D-Trp

D is Ser

E is N-Me-Ala, Tyr, N-Me-Tyr, Ser, Lys(iPr), 4-Cl-Phe, His, Asn, Met,Ala, Arg or Ile;

F is ##STR11## wherein R and X are, independently, H or alkyl; and

T comprises a receptor-modifying moiety;

G is Leu or Trp;

H is Lys(iPr), Gln, Met, or Arg

I is Pro; and

J is Gly-NH₂ or D-Ala-NH₂ ;

or a pharmaceutically acceptable salt thereof.

In preferred embodiments, T is selected from the group consisting ofylids, sulfonium moieties, α-halocarbonyls, sulfates, sulfonates, alkylhalides and benzyl halides. In a particularly preferred embodiment, T isan α-halocarbonyl.

In another embodiment, the invention provides a peptide comprising astructure:

    A-B-C-D-E-F-G-H-I-J                                        (SEQ ID NO: 5)

wherein

A is pyro-Glu, Ac-D-Nal, Ac-D-Qal, Ac-Sar, or Ac-D-Pal

B is His or 4-Cl-D-Phe

C is Trp, D-Pal, D-Nal, L-Nal, D-Pal(N-O), or D-Trp

D is Ser

E is N-Me-Ala, Tyr, N-Me-Tyr, Ser, Lys(iPr), 4-Cl-Phe, His, Asn, Met,Ala, Arg or Ile;

F is ##STR12## wherein R and X are, independently, H or alkyl; and

M comprises an N-acyl hydrophilic moiety;

G is Leu or Trp;

H is Lys(iPr), Gln, Met, or Arg

I is Pro; and

J is Gly-NH₂ or D-Ala-NH₂ ;

or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a peptide comprising astructure:

    A-B-C-D-E-F-G-H-I-J                                        (SEQ ID NO: 6)

wherein

A is pyro-Glu, Ac-D-Nal, Ac-D-Qal, Ac-Sar, or Ac-D-Pal

B is His or 4-Cl-D-Phe

C is Trp, D-Pal, D-Nal, L-Nal, D-Pal(N-O), or D-Trp

D is Ser

E is N-Me-Ala, Tyr, N-Me-Tyr, Ser, Lys(iPr), 4-Cl-Phe, His, Asn, Met,Ala, Arg or Ile;

F is ##STR13## wherein R and X are, independently, H or alkyl; and

L comprises a small polar moiety;

G is Leu or Trp;

H is Lys(iPr), Gln, Met, or Arg

I is Pro; and

J is Gly-NH₂ or D-Ala-NH₂ ;

or a pharmaceutically acceptable salt thereof.

In preferred embodiments, F is selected from the group consisting ofD-Cit, D-Asn, D-Gln, and D-Thr. In a particularly preferred embodiment,the peptide comprises a structure:

    Ac-D-Nal-4-Cl-D-Phe-D-Pal-Ser-N-Me-Tyr-D-Asn-Leu-Lys(iPr)-Pro-Ala-NH.sub.2;

or a pharmaceutically acceptable salt thereof.

In another particularly preferred embodiment, the peptide comprising astructure:

    Ac-D-Nal-4-Cl-D-Phe-D-Pal-Ser-Tyr-D-Asn-Leu-Lys(iPr)-Pro-D-Ala-NH.sub.2 ;

or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides an LHRH antagonist, comprisinga peptide having a sidechain modified by a dipolar moiety forming amodified peptide, such that the modified peptide has FHRH antagonistactivity. In preferred embodiments, the dipolar moiety is selected fromthe group consisting of ylids, tertiary amine oxides, nitrile oxides,pyridine-N-oxides, and pyridinium zwitterions. In more preferredembodiments, the dipolar moiety is an ylid, a pyridine-N-oxide or apyridinium zwitterion. In other preferred embodiments, the peptidecomprises about 8 to about 12 residues. In more preferred embodiments,the peptide comprises 10 residues. In certain preferred embodiments, thedipolar moiety modifies residue 6. In certain preferred embodiments, theLHRH antagonist is a peptide mimetic.

In another embodiment, the invention provides an LHRH antagonist,comprising a peptide having a sidechain modified by a cationic moietyselected from the group consisting of cationic pyridinium moieties andsulfonium moieties, with the proviso that the cationic moiety is notN-methyl pyridinium, forming a modified peptide, such that the modifiedpeptide has LHRH antagonist activity. In preferred embodiments, thecationic moiety is a cationic pyridinium moiety. In other preferredembodiments, the cationic moiety is a sulfonium moiety. In otherpreferred embodiments, the peptide comprises about 8 to about 12residues. In more preferred embodiments, the peptide comprises 10residues. In other preferred embodiments, the cationic moiety modifiesat least one of residue 6 and residue 8. In other preferred embodiments,the LHRH antagonist is a peptide mimetic.

In another embodiment, the invention provides an LHRH antagonist,comprising a peptide having a sidechain modified by a receptor-modifyingmoiety forming a modified peptide, such that the modified peptide hasLHRH antagonist activity. In preferred embodiments, thereceptor-modifying moiety is selected from the group consisting ofylids, sulfonium moieties, α-halocarbonyls, sulfates, sulfonates alkylhalides, and benzyl halides. In preferred embodiments, the peptidecomprises about 8 to 12 residues. In more preferred embodiments, thepeptide comprises 10 residues. In preferred embodiments, thereceptor-modifying moiety modifies residue 6. In preferred embodiments,the LHRH antagonist is a peptide mimetic.

In another embodiment, the invention provides an LHRH antagonist,comprising a peptide having a sidechain modified by a hydrophilic N-acylmoiety forming a modified peptide, such that the modified peptide hasLHRH antagonist activity. In preferred embodiments, the hydrophilicN-acyl moiety modifies position 6. In preferred embodiments, a residuecomprises a hydrophilic acyl moiety is selected from the groupconsisting of D-Lys(Imdac), D-Lys(Ppic), and D-Lys(Dodac). In preferredembodiments, the hydrophilic N-acyl moiety has a log P between -1 and+2.

In another embodiment, the invention provides an LHRH antagonist,comprising a peptide having a small polar moiety in position 6, suchthat the peptide has LHRH antagonist activity. In preferred embodiments,the antagonist has an AOA less than about 1 μg. In preferredembodiments, the antagonist has a histamine-releasing activity of atleast about 5 μg.

II. Pharmaceutical Compositions

The LHRH antagonist peptides of the invention can be incorporated intopharmaceutical compositions suitable for administration to a subject. Ina preferred embodiment, the pharmaceutical composition comprises an LHRHantagonist peptide of the invention and a pharmaceutically acceptablecarrier.

A "therapeutically effective amount" refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredresult. A therapeutically effective amount of an LHRH antagonist peptideof the present invention may vary according to factors such as thedisease state, age, sex, and weight of the individual, and the abilityof the antagonist to elicit a desired response in the individual. Dosageregimens may be adjusted to provide the optimum therapeutic response. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antagonist are outweighed by thetherapeutically beneficial effects. A non-limiting range for atherapeutically effective amount of an LHRH antagonist is 0.01 μg/kg-10mg/kg, preferably between about 0.01 and 5 mg/kg. It is to be noted thatdosage values may vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

As used herein, "pharmaceutically acceptable carrier" includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous or parenteral administration(e.g., by injection). Depending on the route of administration, theactive compound may be coated in a material to protect the compound fromthe action of acids and other natural conditions which may inactivatethe compound.

A "pharmaceutically acceptable salt" refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects. Examples of such salts are salts ofacids such as hydrochloric acid, hydrobromic acid, sulfuric acid,phosporic acid, nitric acid, and the like; acetic acid, oxalic acid,tartaric acid, succinic acid, malic acid, benzoic acid, pamoic acid,alginic acid, methanesulfonic acid, naphthalenesulfonic acid, and thelike. Also included are salts of cations such as sodium, potassium,lithium, zinc, copper, barium, bismuth, calcium, and the like; ororganic cations such as trialkylammonium. Combinations of the abovesalts are also useful.

The LHRH antagonists of the present invention can be administered by avariety of methods known in the art. In a preferred embodiment, the LHRHantagonists are administered in a time release formulation, for examplein a composition which includes a slow release polymer, or by depotinjection. The active compounds can be prepared with carriers that willprotect the compound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

The LHRH antagonists can be orally administered, for example, with aninert diluent or an assimilable edible carrier. The LHRH antagonists andother ingredients may also be enclosed in a hard or soft shell gelatincapsule, compressed into tablets, or incorporated directly into thesubject's diet. For oral therapeutic administration, the LHRHantagonists may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of the LHRHantagonists in the compositions and preparations may, of course, bevaried. The amount of the LHRH antagonists in such therapeuticallyuseful compositions is such that a suitable dosage will be obtained.

To administer the LHRH antagonists by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the LHRH antagonists may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al., (1984) J. Neuroimmunol.71:27). Pharmaceutically acceptable carriers include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersion. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the pharmaceuticalcompositions of the invention is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmanitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., LHRH antagonist) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Dosage regimens may be adjusted to provide the optimum therapeuticresponse. For example, a single bolus may be administered, severaldivided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

III. Methods of Using the LHRH Antagonists of the Invention

The LHRH antagonist peptides of the present invention are useful for thetreatment of such conditions as precocious puberty, prostate cancer,ovarian cancer, benign prostatic hypertrophy, endometriosis, uterinefibroids, breast cancer, premenstrual syndrome, polycystic ovarysyndrome, and diseases which result from excesses of gonadal hormones inhumans or animals of either sex. The LHRH antagonist peptides of theinvention are also useful for behavior modification (e.g., "chemicalcastration"). The LHRH antagonist peptides are also useful forcontrolling reproduction in both males and females. Furthermore, thepeptides of the invention may be used to treat immunosuppressedpatients, as described in, for example, U.S. Pat. No. 5,003,011.

Thus, in one embodiment, the invention provides a method of inhibitingLHRH activity in a subject, comprising administering to a subject aneffective amount of an LHRH antagonist of the present invention, suchthat LHRH activity is inhibited.

In another embodiment, the invention provides a method of inhibitingLHRH activity in a cell, comprising contacting a cell with an LHRHantagonist of the invention, such that LHRH activity is inhibited.

In another embodiment, the invention provides a method of inhibitinggrowth of a hormone-dependent tumor in a subject, comprisingadministering to a subject an effective amount of an LHRH antagonist ofthe invention, such that tumor growth is inhibited.

In another embodiment, the invention provides a method of inhibitingovulation in a subject, comprising administering to a subject aneffective amount of an LHRH antagonist of the invention, such thatovulation is inhibited.

In another aspect, the invention provides a packaged formulation fortreating a subject for a disorder associated with LHRH activity,comprising an LHRH antagonist of the invention packaged withinstructions for using the LHRH antagonist for treating a subject havinga disorder associated with LHRH activity.

Exemplification

In the examples, the following abbreviations are used:

Boc: N-t-butoxycarbonyl

HOBt: 1-hydroxybenzotriazole

MCPBA: m-chloroperbenzoic acid

DCC: dicyclohexylcarbodiimide

Anti-ovulatory activity (AOA) was measured by an in vivo assay in rats,as described in Corbin and Beattie, Endocrine Res. Commun. 2:1 (1975).In brief, female rats are injected with a candidate LHRH antagonist onthe day of proestrus; in general, the candidate LHRH antagonist wasdissolved in 0.1% DMSO. The ability of the candidate peptide to inhibitovulation is measured by determining the number of rats which ovulate. Acandidate peptide is considered to have LHRH antagonist qualities if itinhibits ovulation in at least 50% of the treated rats at a dose of 5 μgper rat. Preferred LHRH antagonists inhibit ovulation in at least 50% ofrats at a dose of 2 μg per rat, more preferably at a dose of 1 μg perrat, and still more preferably at a dose of 0.5 μg per rat.

Histamine-releasing activity was assayed by the method described in U.S.Pat. No. 4,851,385 to Roeske. Briefly, a suspension of rat mast cellswas added to increasing concentrations of an LHRH antagonist peptide andincubated for 15 minutes at 37° C. The buffer contained 25 mM PIPES, pH7.4, NaCl (119 mM), KCl (5 mM), NaOH (40 mM) glucose (5.6 mM), CaCl₂ (1mM) and 0.1% bovine serum albumin. The reaction was stopped bycentrifugation at 400× g for 15 minutes at 4° C., and the supernatantassayed for histamine content by a published method (Siriganian (1974)Anal. Biochem. 57:383 and Siriganian and Hook (1986) in "Manual ofClinical Immunology", 3rd ed., N. R. Rose, H. Friedman, and J. L Fahey,eds., p. 808), or by a manual method which gave similar results. Maximalhistamine release occurred rapidly, typically in less than one minute.No evidence of cell toxicity was seen for any of the peptides tested.The histamine-releasing activity of peptides is measured as the ED₅₀, inμg/ml; a higher ED₅₀ represents lower histamine release.

The AOA and histamine-releasing activities of several peptides aresummarized in Table 1.

                  TABLE I                                                         ______________________________________                                        LHRH Antagonists                                                                                                   Hista-                                   Com-                       AO activity                                                                             mine                                     pound Sequence             (AOA)     release                                  ______________________________________                                        3341  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              4/10 @ 1 μg                                                                          106                                            D--Pal.sup.3, D--Gln.sup.6--Lys(iPr).sup.8,                                                        2/10 @ 2 μg                                           D--Ala.sup.10 -LHRH.TFA                                                 3342  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              2/10 @ 2 μg                                                                          126                                            D--Pal.sup.3, D--Asn.sup.6 --Lys(iPr).sup.8,                                  D--Ala.sup.10 -LHRH.TFA                                                 3343  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              5/10 @ 1 μg                                                                           62                                            D--Pal.sup.3, D--Thr.sup.6 --Lys(iPr).sup.8,                                  D--Ala.sup.10 --LHRH.TFA                                                3344  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              9/10 @ 1 μg                                                                           32                                            D--Pal.sup.3, D--Cit.sup.6 --Lys(iPr).sup.8,                                  Pip.sup.9 --D--Ala.sup.10 -LHRH.TFA                                     3361  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              1/8 @ 5 μg                                                                           131                                            D--Pal.sup.3, D--Glu(Taurine).sup.6 -Lys(iPr).sup.8,                          D--Ala.sup.10 -LHRH.TFA                                                 3362  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              4/8 @ 1 μg                                                                            22                                            D--Pal.sup.3,                                                                 D--Cit.sup.6 --Lys(iPr).sup.8 -LHRH.TFA                                 3363  Ac--D--Nal.sup.1, 4-Cl-D--Phe.sup.2,                                                               6/8 @ 1 μg                                                                            25                                            D--Pal.sup.3, D--Cit.sup.6 --Lys(iPr).sup.8,                                  Pip.sup.9 -LHRH.TFA                                                     3364  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1 μg                                                                            14                                            D--Pal.sup.3, D--Phe(4-NO.sub.2).sup.6 --Lys(iPr).sup.8,                      D--Ala.sup.10 -LHRH.TFA                                                 3365  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                                         24                                            D--Pal.sup.3, D--Cit.sup.6 --Lys(iPr).sup.8,                                  ProNHEt.sup.9 -des-Gly.sup.10 -LHRH.TFA                                 3366  Ac--D-(or L)-9-anthryl-Ala.sup.1,                                                                  6/8 @ 5 μg                                            4-Cl--D--Phe.sup.2, D--Pal.sup.3,                                             D--Cit.sup.6 --Lys(iPr).sup.8,                                                D--Ala.sup.10 -LHRH.TFA                                                 3367  Ac-L-(or D)-9-anthryl-Ala.sup.1,                                                                   7/8 @ 5 μg                                            4-Cl--D--Phe.sup.2, D--Pal.sup.3,                                             D--Cit.sup.6 --Lys(iPr).sup.3,                                                D--Ala.sup.10 -LHRH.TFA                                                 3368  Ac--D-(or L)-Ada--Ala.sup.1,                                                                       5/8 @ 5 μg                                                                            34                                            4-Cl--D--Phe.sup.2, D--Pal.sup.3,                                             D--Cit.sup.6 --Lys(iPr).sup.8,                                                D--Ala.sup.10 -LHRH.TFA                                                 3369  Ac--L-(or D)-Ada--Ala.sup.1,                                                                       7/8 @ 5 μg                                                                            93                                            4-Cl--D--Phe.sup.2, D--Pal.sup.3,                                             D--Cit.sup.6 --Lys(iPr).sup.8,                                                D--Ala.sup.10 -LHRH.TFA                                                 3423A Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/10 @     52                                            D--Pal.sup.3, D--Lys(Glc).sup.6 --Lys(iPr).sup.8,                                                  0.5 μg                                                D--Ala.sup.10 -LHRH.TFA                                                                            2/10 @ 1 μg                                                                0/10 @ 2 μg                                     3428  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              1/8 @ 1 μg                                            D--Pal.sup.3, D--Pal(1-Bu).sup.6, Lys(iPr).sup.8,                             D--Ala.sup.10 -LHRH.TFA                                                 3429  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              0/8 @ 1 μg                                                                           6.0                                            D--Pal.sup.3, D--Pal(Bzl).sup.6, Lys(iPr).sup.8,                                                   4/8 @ 0.5 μg                                          D--Ala.sup.10 -LHRH.TFA                                                 3430  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              3/8 @ 1 μg                                                                            10                                            D--Pal.sup.3, Pal.sup.5, D--Pal(iPr).sup.6,                                   Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3431  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1 μg                                            D--Pal.sup.3, Cit.sup.5, D--Cit.sup.6,                                        Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3432  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 @ 1 μg                                            D--Pal.sup.3, Pal.sup.5, D--Cit.sup.6,                                        Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3433  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              5/8 @ 1 μg                                            D--Pal.sup.3, Cit.sup.5, D--Pal.sup.6,                                        Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3434  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              4/8 @ 1 μg                                            D--Pal.sup.3, Asn.sup.4, Tyr.sup.5,                                           D--Cit.sup.6, Lys(iPr).sup.8,                                                 D--Ala.sup.10 -LHRH.TFA                                                 3435  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 @ 1 μg                                            D--Pal.sup.3, Cit.sup.5, D--Pal(iPr).sup.6,                                   Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3436  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 @ 1 μg                                            D--Pal.sup.3, D--HomoArg(NO.sub.2).sub.6,                                     Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3437  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 @ 1 μg                                            D--Pal.sup.3, D--Lys(Glycolyl).sup.6,                                         Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3438  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              2/8 @ 1 μg                                                                           5.6                                            D--Pal.sup.3, D--Lys(iPrPic).sup.6,                                           Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3439  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              2/8 @ 1 μg                                            D--Pal.sup.3, D--Lys(HomoPro).sup.6,                                          Lys(iPr)8, D--Ala.sup.10 -LHRH.TFA                                      2958  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, D--Pal(iPr).sup.6,                                              Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3440  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              2/8 @ 1 μg                                            D--Pal.sup.3, D--Lys(3-pyridineacetic).sup.6,                                 Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3441  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              8/8 @ 1 μg                                            D--Pal.sup.3, D--Lys(2-ClNic).sup.6,                                          Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3442  Ac--N.sup.α Me--D--Nal.sup.1,                                                                7/8 @ 1 μg                                            4-Cl--D--Phe.sup.2, D--Pal.sup.3, D--Cit.sup.6,                               Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3502  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1 μg                                            D--Pal.sup.3, Lys(Otac).sup.5, D--Lys(Otac).sup.6,                            Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3503  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1 μg                                            D--Pal.sup.3, Lys(ONic).sup.5, D--                                            Lys(ONic).sup.6, Lys(iPr).sup.8,                                              D--Ala.sup.10 -LHRH.TFA                                                 3504  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              4/8 @ 1μg                                             D--Pal.sup.3, Lys(Pyz).sup.5, D--Lys(Pyz).sup.6,                              Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3552  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              8/8 @ 1 μg                                            D--Pal.sup.3, D--Glu.sup.6, Lys(iPr).sup.8,                                   D--Ala.sup.10 -LHRH.TFA                                                 3505  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              5/8 @ 1 μg                                            D--Pal.sup.3, D--Lys.sup.6, Lys(iPr).sup.8,                                   D--Ala.sup.10 -LHRH.TFA                                                 3506  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              4/8 @ 1 μg                                            D--Pal.sup.3, D--Lys(Gulonyl).sup.6,                                          Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3553  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              8/8 @ 1 μg                                            D--Pal.sup.3, D--Lys(iPrNic).sup.5,                                           D--Lys(iPrNic).sup.6, Lys(iPr).sup.8,                                         D--Ala.sup.10 -LHRH.TFA                                                 3507  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              1/8 @ 1 μg                                            D--Pal.sup.3, D--Lys(ONic).sup.6                                              Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3508  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              0/8 @ 1 μg                                            D--Pal.sup.3, D--Lys(OTac).sup.6                                                                   6/8 @ 0.5 μg                                          Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3509  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, D--Lys(Pyz).sup.6                                               Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3510  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, D--Lys(nBuNic).sup.6,                                           Lys(iPr)8, D--Ala.sup.10 -LHRH.TFA                                      3511  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 @ 1 μg                                            D--Pal.sup.3, D--Glu(Amp).sup.6,                                              Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3543  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1 μg                                            D--Pal.sup.3, D--Glu(Dea).sup.6,                                              Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3563  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, Lys(pGlu).sup.5,                                                D--Lys--Glu).sup.6, Lys(iPr).sup.8,                                           D--Ala.sup.10 -LHRH.TFA                                                 3540  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, N.sup.α Me--Tyr.sup.5,                                    D--Pal(iPr).sup.6, Lys(iPr).sup.8,                                            D--Ala.sup.10 -LHRH.TFA                                                 3541  Ac--D--Nal.sup.1, C.sup.α Me--4Cl--Phe.sup.2,                                                1/8 @ 1 μg                                            D--Pal.sup.3, N.sup.α Me-Tyr.sup.5,                                     D--Pal(iPr).sup.6, Lys(iPr).sup.8,                                            D--Ala.sup.10 -LHRH.TFA                                                 3554  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, Lys(CNa).sup.6,                                                 Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3542  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 @ 2 μg                                            D--Pal.sup.3, D--Glu(PEG).sup.6,                                              Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3565  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, D--Lys(Oxa).sup.6,                                              Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3551  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1 μg                                            D--Pal.sup.3, Lys(CNa).sup.5, D--Lys(CNa).sup.6,                              Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3544  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1 μg                                            D--Pal.sup.3, Lys(ClNic).sup.5,                                               D--Lys(ClNic).sup.6, Lys(iPr).sup.8,                                          D--Ala.sup.10 -LHRH.2TFA                                                3555A Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, D--Lys(Ac).sup.6,                                               Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3564  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, D--Glu(Tris).sup.6,                                             Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3545  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 @ 1 μg                                            D--Pal.sup.3, D--Gln(iPr).sup.6,                                              Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3550  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1 μg                                            D--Pal.sup.3, D--Glu(CSer).sup.6,                                             Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3549  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1 μg                                            D--Pal.sup.3, D--Glu(Mop).sup.6,                                              Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3548  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1 μg                                            D--Pal.sup.3, Lys.sup.5, D--Pal(iPr).sup.6,                                   Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.4TFA                                3566  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, Lys(Nic).sup.5,                                                 D--Pal(iPr).sup.6, Lys(iPr).sup.8,                                            D--Ala.sup.10 -LHRH.3TFA                                                3567  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, Lys(Ac).sup.5, D--Pal(iPr).sup.6,                               Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.3TFA                                3568  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, D--Glu(DEGA).sup.6,                                             Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3547  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1 μg                                            D--Pal.sup.3, Lys(Nic).sup.5,                                                 D--Pal(Bzl).sup.6, Lys(iPr).sup.8,                                            D--Ala.sup.10 -LHRH.3TFA                                                3569  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, Lys(Ac).sup.5, D--Pal(Bzl).sup.6,                               Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.3TFA                                3546  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1 μg                                            D--Pal.sup.3, Lys(TFAc).sup.6, Lys(iPr).sup.8,                                D--Ala.sup.10 -LHRH.2TFA                                                3570  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, Lys.sup.5, D--Pal(Bzl).sup.6,                                   Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.3TFA                                3571  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, Lys(iPr).sup.5, D--Lys(Nic).sup.6,                              Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.3TFA                                3572  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, Lys(iPr).sup.5,                                                 D--Lys(Pic).sup.6, Lys(iPr).sup.8,                                            D--Ala.sup.10 -LHRH.3TFA                                                      Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, Lys(TFAc).sup.5,                                                D--Lys(TFAc).sup.6, Lys(iPr).sup.8,                                           D--Ala.sup.10 -LHRH.2TFA                                                      Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, Lys(iPr).sup.5,                                                 4-Cl--D--Phe.sup.6, Lys(iPr).sup.8,                                           D--Ala.sup.10 -LHRH.3TFA                                                      Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, Lys(iPr).sup.5,                                                 D--Nal.sup.6, Lys(iPr).sup.8,                                                 D--Ala.sup.10 -LHRH.3TFA                                                      Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, Lys(iPr).sup.5,                                                 D--Lys(pGlu).sup.6, Lys(iPr).sup.8,                                           D--Ala.sup.10 -LHRH.3TFA                                                      Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, Lys(iPr).sup.5,                                                 D--Lys(OTac).sup.6, Lys(iPr).sup.8,                                           D--Ala.sup.10 -LHRH.3TFA                                                      Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, Ile.sup.5,                                                      D--Pal(Bzl).sup.6, Lys(iPr).sup.8,                                            D--Ala.sup.10 -LHRH.3TFA                                                      Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Pal.sup.3, Lys(Pic).sup.5,                                                 D--Pal(iPr).sup.6, Lys(iPr).sup.8,                                            D--Ala.sup.10 -LHRH.3TFA                                                3721  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              4/8 @ 1.0                                                D--Pal.sup.3, D--Lys(3-Δ-Pro).sup.6,                                    Lys(iPr).sup.8, D--Ala.sup.10 -LHRH--TFA                                3722  Ac--D--Nal-4-Cl--D--Phe.sup.2, D--Pal.sup.3,                                                       8/8 @ 5.0                                                D--Lys(Ac--D--Nal.sup.1, 4-                                                   Cl--D--Phe.sup.2, D--Pal.sup.3, Ser).sup.6,                                   Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3723  Ac--D--Qal.sup.1,4-Cl--D--Phe.sup.2,                                                               7/8 @ 1.0                                                D--Pal.sup.3, D--Lys(pGlu).sup.6,                                             Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3740  Ac--D--Qal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1.0                                                D--Pal.sup.3, D--Lys(Ac).sup.6,                                               Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3741  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              1/8 @ 1.0                                                D--Pal.sup.3, D--Lys(Imdac).sup.6, Lys(iPr).sup.8,                                                 6/8 @ 0.5                                                D--Ala.sup.10 -LHRH.TFA                                                 3742  Ac--D--Qal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1.0                                                D--Pal.sup.3, D--Lys(Dodac).sup.6, Lys(iPr).sup.8,                            D--Ala.sup.10 -LHRH.TFA                                                 3743  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              8/8 @ 1.0                                                D--Pal.sup.3, Ser.sup.5, D--Pal(iPr).sup.6,                                   Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3753  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 @ 1.0                                                D--Pal.sup.3, D--Lys(iPr).sup.5,                                              D--Lys(TFAc).sup.6, Lys(iPr).sup.8,                                           D--Ala.sup.10 -LHRH.2TFA                                                3754  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              5/8 @ 1.0                                                D--Pal.sup.3, His.sup.5 D--Pal(iPr).sup.6,                                    Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3744  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 @ 1.0                                                D--Pal.sup.3, Asn.sup.5, D--Pal(iPr).sup.6,                                   Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3745  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1.0                                                D--Pal.sup.3, Lys(iPr).sup.5, D--Lys(4HBc).sup.6,                             Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3755  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 2 1.0                                                D--Pal.sup.3, Met.sup.5, D--Pal(iPr).sup.6,                                   Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3756  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1.0                                                D--Phe.sup.2, D--Pal.sup.3, Ala.sup.5, D--Pal(iPr).sup.6,                     Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3757  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 @ 1.0                                                D--Pal.sup.3, N--Me--Ala.sup.5, D--Pal(iPr).sup.6,                            Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3758  Ac--D--NaI1 , 4-Cl--D--Phe.sup.2,                                                                  5/8 @ 1.0                                                D--Pal.sup.3, D--Lys(Hippic).sup.6,                                           Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                       D--Pal.sup.3, D--Lys(Hippic).sup.6,                                     3759  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              5/8 @ 1.0                                                D--Pal.sup.3, D--Lys(AcGly).sup.6,                                            Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3760  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              1/8 @ 1.0                                                D--Pal.sup.3, D--Lys(Ppic).sup.6,                                                                  7/8 @ 0.5                                                Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3761  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 @ 1.0                                                D--Pal.sup.3, D--Lys(Mts).sup.6,                                              Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3762  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1.0                                                D--Pal.sup.3, D--Lys(Orotic).sup.6,                                           Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3763  Ac--Sar.sup.1, 4-Cl--D--Phe.sup.2,                                                                 4/8 @ 1.0 102.sup.1,                                     D--Nal.sup.3, D--Pal(Bzl).sup.6, Lys(iPr).sup.8,                                                             76.sup.b                                       D--Ala.sup.10 -LHRH.2TFA                                                3769  Ac--Sar.sup.1, 4-Cl--D--Phe.sup.2, 1-1-Nal.sup.3,                                                  6/8 @ 1.0                                                D--Pal(Bzl).sup.6, Lys(iPr).sup.8,                                            D--Ala.sup.10 -LHRH.2TFA                                                3770  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1.0                                                D--Pal.sup.3, D--Pal(CH.sub.2 COOH).sup.6,                                                         0.1 DMSO                                                 Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3771  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 @ 1.0                                                D--Pal.sup.3, D--Lys(Ala).sup.6,                                              Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3772  Ac--Sar.sup.1, 4-Cl--D--Phe.sup.2,                                                                 8/8 @ 1.0                                                D-1-Nal.sup.3, D--Pal(iPr).sup.6,                                             Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3773  Ac--Sar.sup.1, 4-Cl--D--Phe.sup.2, L--Nal.sup.3,                                                   7/8 @ 1.0                                                D--Pal(iPr).sup.6, Lys(iPr).sup.8,                                            D--Ala.sup.10 -LHRH.2TFA                                                3785  Ac--D--Qal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 @ 1.0                                                D--Pal.sup.3, D--Lys(Gulonyl).sup.6, Lys(iPr).sup.8,                                               0.1 DMSO                                                 D--Ala.sup.10 -LHRH.TFA                                                 3786  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              4/8 @ 1.0                                                D--Pal.sup.3, D--Pal(N--O).sup.6,                                             Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3787  Ac--D--Qal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 @ 1.0                                                D--Pal.sup.3, D--Lys(Ppic).sup.6,                                             Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3800  Ac--D--Qal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 @ 1.0                                                D--Pal.sup.3, D--Lys(Imdac).sup.6,                                            Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3801  Ac--D--Qal.sup.1, 4-Cl--D--Phe.sup.2,                                                              8/8 @ 1.0                                                D--Pal.sup.3, D--Lys(Onic).sup.6,                                             Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3802  Ac--D--Qal.sup.1, 4-Cl--D--Phe.sup.2,                                                              5/8 @ 1.0                                                D--Pal.sup.3, D--Lys(Otac).sup.6,                                             Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3803  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              2/8 @ 1.0                                                D--Pal.sup.3, D--Lys(Dodac).sup.6,                                            Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3804  Ac--D--Pal.sup.1, 4-Cl--D--Phe.sup.2,                                                              8/8 @ 1.0                                                D--Pal.sup.3, D--Pal(iPr).sup.6,                                              Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3827  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              4/8 @ 1.0                                                D--Pal.sup.3, N--Me--Tyr.sup.5, D--Asn.sup.6,                                 Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3828  Ac--D--Na1.sup.1, 4-Cl--D--Phe.sup.2,                                                              4/8 @ 1.0 42.sup.a,                                      D--Pal.sup.3, N--Me--Tyr.sup.5,                                                                              39.sup.b                                       D--Lys(Oric).sup.8, Lys(iPr).sup.8,                                           D--Ala.sup.10 -LHRH.TFA                                                 3829  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              8/8 @ 1.0                                                D--Pal.sup.3, N--Me--Tyr.sup.5, D--Lys(Ac).sup.6,                             Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.TFA                                 3852  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              8/8 @ 5.0                                                D--Pal.sup.3, Lys(iPr).sup.5, D--His.sup.6,                                   Trp.sup.7, Orn.sup.8, D--Ala.sup.10 -LHRH.TFA                           3853  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              0/8 @ 5.0                                                D--Pal.sup.3, His.sup.5, D--Arg.sup.6,                                        Trp.sup.7, Orn.sup.8, D--Ala.sup.10 -LHRH.TFA                           3854  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 5.0                                                D--Pal.sup.3, Arg.sup.5, D--His.sup.6,                                        Trp.sup.7, Orn.sup.8, D--Ala.sup.10 -LHRH.TFA                           3855  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              4/8 @ 1.0                                                D--Pal.sup.3, Lys(iPr).sup.5, D--Trp.sup.6,                                   Trp.sup.7, Orn.sup.8, D--Ala.sup.10 -LHRH.TFA                           3851  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              0/8 @ 1.0 4.1.sup.a,                                     D--Pal.sup.3, 4-Cl--Phe.sup.5, D--Pal(iPr).sup.6,                                                  6/8 @ 0.5 5.4.sup.b                                      Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3882  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1.0                                                D--Pal(N--O).sup.3, D--Pal(iPr).sup.6,                                        Lys(iPr).sup.8, D--Ala.sup.10 -LHRH.2TFA                                3880  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              8/8 @ 1.0                                                D--Trp.sup.3, D--Arg.sup.6, Met.sup.8,                                        D--Ala.sup.10 -LHRH.TFA                                                 3878  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              2.8 @ 1.0                                                D--Trp.sup.3, D--Arg.sup.6, Met(S.sup.+ Me).sup.8,                                                 7/8 @ 0.5                                                D--Ala.sup.10 -LHRH.2TFA                                                3881  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              7/8 @ 1.0                                                D--Trp.sup.3, D--Met.sup.6,                                                   D--Ala.sup.10 -LHRH.TFA                                                 3879  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              0/8 @ 1.0                                                D--Trp.sup.3, D--Met(S.sup.+ Me).sup.6,                                                            6/8 @ 0.5                                                D--Ala.sup.10 -LHRH.2TFA                                                3926  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1.0                                                D--Trp.sup.3, D--Arg.sup.6, Lys(COCH.sub.2 Br).sup.8,                         D--Ala.sup.10 -LHRH.TFA                                                 3925  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              5/8 @ 1.0 13.sup.c,                                      D--Trp.sup.3, D--Met(S.sup.+ Me).sup.6,                                                                      5.sup.d                                        Met(S.sup.+ Me).sup.8, D--Ala.sup.10 -LHRH.2TFA                         3941  Met(S.sup.+ Me).sup.8 -LHRH.TFA                                                                    8/8 @ 50.0                                         3942  Lys(COCH.sub.2 Br).sup.8 -LHRH                                                                     8/8 @ 50.0                                         3948  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              low activity                                             D--Trp.sup.3, D--Lys(COCH.sub.2 Br).sup.6,                                    D--Ala.sup.10 -LHRH.TFA                                                 3949* Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              low activity                                             D--Pal.sup.3, D--Lys(COCH.sub.2 CH.sub.2 N(Et).sub.2).sup.6,                  D--Ala.sup.10 -LHRH.2TFA                                                3960  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              5/8 @ 1.00                                               D--Pal.sup.3, D--Lys(2-pyrimidyl-                                             thio)acetic).sup.6, Lys(iPr).sup.8,                                           D--Ala.sup.10 -LHRH.TFA                                                 3961  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              4/8 @ 2.00                                               D--Trp.sup.3, D--Met(S.sup.+ CH.sub.2 C.sub.6 H.sub.5).sup.6,                                      3/8 @ 1.00                                               D--Ala.sup.10 -LHRH.2TFA                                                3967  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              1/8 @ 0. 5                                                                              0.38.sup.e,                                    D--Pal.sup.3, D--Met(S.sup.+ CH.sub.3).sup.6,                                                      7/8 @ 0.25                                                                              0.15.sup.f                                     D--Ala.sup.10 -LHRH.2TFA                                                3968* Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              4/8 @ 1.00                                               D--Pal.sup.3, D--Met(S.sup.+ CH.sub.2 COPh).sup.6,                            D--Ala.sup.10 -LHRH.2TFA                                                3969  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              3/8 @ 1.00                                               D--Pal.sup.3, D--Dap(COCH.sub.2 S.sup.+ Me.sub.2).sup.6,                      D--Ala.sup.10 -LHRH.2TFA                                                3982  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              8/8 @ 1.00                                                                              53.sup.c,                                      D--Pal.sup.3, His.sup.5 D--Pal(iPr).sup.6, Lys(iPr).sup.8,                                                   33.sup.d                                       D--Ala.sup.10 -LHRH.2TFA                                                3983  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              0/8 @ 1.00                                                                              5.35.sup.g,                                    D--Pal.sup.3, D--Arg.sup.6, Met(S.sup.+ Me).sup.8,                                                 8/8 @ 0.5 1.32.sup.h                                     D--Ala.sup.10 -LHRH.2TFA                                                                           3/8 @ 1.00                                         3984  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                         D--Trp.sup.3,                                                                 D--Met(S.sup.30 CH.sub.2 --CH═CH.sub.2).sup.6,                            D--Ala.sup.10 -LHRH.2TFA                                                3985  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              0/8 @ 1.00                                                                              4.4.sup.a,                                     D--Pal.sup.3, D--Arg.sup.6,                                                                        8/8 @ 0.5 3.86.sup.b                                     Orn(COCH.sub.2 S.sup.+ Me.sub.2).sup.8,                                       D--Ala.sup.10 -LHRH.2TFA                                                3994  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1.00                                               D--Pal.sup.3, D--Arg.sup.6, Orn(COCH.sub.2 SMe).sup.8,                        D--Ala.sup.10 -LHRH.2TFA                                                3995  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              5/8 @ 1.00                                               D--Pal.sup.3, D--Arg.sup.6, Met.sup.8,                                        D--Ala.sup.10 -LHRH.TFA                                                 4014  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              0/8 @ 1.00                                               D--Pal.sup.3, D--Lys(COCH.sub.2 S.sup.+ Me.sub.2).sup.6,                      D--Ala.sup.10 -LHRH.2TFA                                                4015  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              1/8 @ 1.00                                               D--Pal.sup.3, D--Arg.sup.6,                                                   Lys(COCH.sub.2 S.sup.+ Me.sub.2).sup.8,                                       D--Ala.sup.10 -LHRH.2TFA                                                4016  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              6/8 @ 1.00                                               D--Pal.sup.3, D--Met(S.sup.+ Me).sup.6,                                       Met(S.sup.+ Me).sup.8, D--Ala.sup.10 -LHRH.2TFA                         4013  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              0/8 @ 1.00                                               D--Pal.sup.3, D--Orn(COCH.sub.2 S.sup.+ Me.sub.2).sup.6,                      D--Ala.sup.10 -LHRH.2TFA                                                4023  Ac--D--Nal.sup.1, 4-Cl--D--Phe.sup.2,                                                              0/8 @ 1.00                                               D--Pal.sup.3 D--Arg.sup.6,                                                    Dap(COCH.sub.2 S.sup.+ Me.sub.2).sup.8,                                       D--Ala.sup.10 -LHRH.2TFA                                                ______________________________________                                         *= Mass spectral analysis did not confirm the structure, Nal arg              (reference) histamine release values: a = 0.11, b = 0.14, c = 0.28, d =       0.11, e = 0.1, f = 0.02, g = 0.1, h = 0.02                               

Sulfonium moieties confer high AOA on peptides when incorporated atposition 6 (e.g., compound 3879) or position 8 (e.g., compound 3983).Compound 3925 (with two sulfonium moieties) showed low histaminerelease. Both alkyl and α-(sulfonium)carbonyl moieties (e.g., compound4023) are effective. Preliminary experiments with LHRH agonists andantagonists suggest that compounds incorporating a bromoacetyl moietyare bound to the receptor and are not removed by repeated washing.

Certain peptides which have an N-alkyl pyridinium moiety at position 6are unexpectedly active (e.g., compound 3851) in the AOA assay. Somehave very little histamine-releasing activity (e.g., compound 3763)compared to the standard, Nal-Arg. These results are unexpected in lightof the previously reported AOA activity and histamine-releasingqualities of N-methyl pyridinium compounds.

Dipolar moieties generally exhibited modest AOA. Compounds includingLys(Onic)⁶ exhibited favorable qualities; one (compound 3828) showed lowhistamine releasing activity, and compound 3507 showed high AOA.

Several peptides having acylated lysine at position 6 were tested, andshowed good AO activity (e.g. compounds 3741 and 3760).

Compounds having a small polar moiety at position 6 exhibited favorablecombinations of AOA and histamine-releasing activity. For example,compound 3827, which has D-Asn (a small, hydrophilic moiety) at position6 showed moderate AO activity. Compounds 3341, 3342, and 3343 havingD-Gln, D-Asn, and D-Thr, respectively, combined moderate AOA with verylow histamine release. Compound 3361, which had the taurine amide of Gluat position 6, also showed very low histamine release. Compound 3369,which had D-Cit at position 6, also showed low histamine release.

All of the analogs listed in Table 1 can by synthesized by the solidphase method using an automated synthesizer (e.g., Beckman Model 990).The amino acid residues used can be purchased from commercial sources(e.g. Aldrich Chemical Co., Milwaukee, Wis.), or can be produced fromcommercially available starting materials according to known methods.For example, pyridinium-N-oxides can be produced by oxidation of thecorresponding pyridine with, e.g., peroxyacids such as mCPBA (see, e.g.,Example 3, infra). Pyridinium moieties, for example, N-benzyl pyridiniumcompounds, can be produced by N-alkylation of the correspondingpyridine, e.g., by heating in an inert solvent with benzyl bromide (seeExamples 1 and 2, infra). Similarly, sulfonium and phosphonium salts canbe produced by S- or P-alkylation of a sulfide or phosphine,respectively, with an alkylating agent such as, for example, methyliodide. Amino acids which are not obtained commercially can besynthesized in a protected form for coupling, or, if appropriate, can becoupled to form a peptide and subsequently modified to the desired form.

EXAMPLE 1 Synthesis of Boc-D-Pal(Bzl) Hydrobromide Salt

Boc-D-Pal (1.36 g, 6.0 mmol) was suspended in 60 ml of acetonitrile.Benzyl bromide (about 50 mmol) was added and the mixture was warmed to50° C. on a water bath. A clear solution resulted, and was stirred atroom temperature for 16 hours. A white precipitate formed; TLC after 17hours showed some starting remaining starting material; stirringcontinued for a total of 5 days, when the reaction was complete. Thesolvent was evaporated under reduced pressure, and the residuerecrystallized from EtOH/ethyl acetate. Yield: 85%; m.p. 166°-170° C.

EXAMPLE 2 Synthesis of Boc-D-Pal(iPr)

Boc-D-Pal (4.0 g, 17.7 mmol) and Ag₂ O (8.0 g, 34.4 mmol) in 22 ml waterwas stirred at room temperature for 4 hours. The reaction vessel wascooled to 0° C., and 2-iodopropane (20.4 g, 120 mmol) in 40 ml2-propanol was added. After addition was complete, the mixture wasallowed to warm to room temperature and stirred for 4 days. AdditionalAg₂ O (2 g) and 2-iodopropane (2 g) were added after 24 hours and againafter 48 hours. The mixture was filtered, and the precipitate was washedwith ethanol (2×15 ml). The filtrate was evaporated to yield 4.3 g of ayellow oil. Crystallization from ethanol/ethyl acetate gave light yellowcrystals (3.0 g); Yield: 63%; m.p. 182°-185° C.

EXAMPLE 3 Synthesis of Boc-D-Pal(N-O)

Boc-D-Pal (2.0 g, 7.5 mmole) was dissolved in 40 ml acetone and 2.48 g(16.5 mmol) of MCPBA (57-86%; purchased from Aldrich and used asreceived) in 80 ml acetone was added in one portion. The mixture wasstirred at room temperature for 40 hours; a small amount of whiteprecipitate formed as the reaction proceeded. The precipitated wasfiltered and the mother liquor evaporated to yield a white precipitate.The combined solids were washed with ether (to remove chlorobenzoicacid) and recrystallized from ethyl acetate/hexane. Yield: 1.7 g (80%);m.p. 155°-157° C.

EXAMPLE 4

A typical coupling cycle for peptide synthesis with Boc-amino acids on apeptide synthesizer (Beckman Model 990) was as follows:

Methylbenzyhydramine (MBHA) resin (1.18 g, 0.85 meq amino groups/gresin) was weighed into the reaction vessel and washed with two portionsof chloroform (26 ml each). The resin was prewashed with 22% thioanisole(5 ml)/66% trifluoroacetic acid (TFA) in 14 ml dichloromethane (DCM) for5 minutes, and then deprotected for 30 minutes with the samethioanisole/TFA mixture. The resin was washed with three portions ofchloroform (20 ml each), two portions of 2-propanol (26 ml each) and twoportions of DCM (26 ml each). The resin was neutralized with twoportions of 12% diisopropylethylamine (DIPEA) (26 ml each), and thenwashed with four portions of DCM (26 ml each), followed by two portionsof 1:1 DCM:dimethylformamide (DMF) (26 ml each). A solution of aBoc-protected amino acid (2.5 mole equivalents) and HOBt (2.5 moleequivalents) was introduced as a solution in 10 ml DMF, and DCC wasadded (256 mg in 6 DMF). Coupling was allowed to proceed for threehours, or overnight. Hindered residues (e.g., backbone N-methyl aminoacids) required longer coupling times. The resin was washed with two 26ml portions of DMF, followed by two 26 ml portions of 2-propanol andthen two 26 ml portions of DCM. Completion of coupling was assessed byKaiser's test (ninhydrin test). If coupling is not complete, a doublecoupling was performed (i.e., the resin was neutralized as above and thecoupling step repeated). When complete coupling is achieved, the cyclewas repeated with the next amino acid.

Upon completion of the synthesis, the peptide was cleaved from the resinby treatment with liquid hydrofluoric acid (HF) for 45 minutes at 0° C.The HF was evaporated and the peptide treated with aqueous acetic acidand lyophilized. The crude peptide was then purified by high performanceliquid chromatography (HPLC) on a C₁₈ column, eluting with a mixture ofacetonitrile and 0.1% TFA in water. Purified fractions (homogeneous byUV and TLC analysis) were combined and lyophilized. Analytical HPLC wasused to determine the purity of the final product; all peptidessynthesized were at least 98% pure.

The contents of all references cited throughout this patent applicationare hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GluHisTrpSerTyrGlyLeuArgProGly                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= Xaa is pyroglutamyl or                          acetylsarcosine                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= Xaa is Trp or                                   3-(2- naphthyl)alaninyl                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= Xaa is N-methyl-Ala, Tyr,                       N-methyl- Tyr, Ser, N-e-2-propyl-lysinyl,                                     (4'-chlorophenyl)alaninyl, His, Asn, Met, Ala, Arg or Ile                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= Xaa is an amino acid having a                   side chain comprising a dipolar moiety                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= Xaa is Leu or Trp                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= Xaa is N-e-2-propyl-lysinyl,                    Gln, Met or Arg                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= Xaa is Gly-NH2                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       XaaHisXaaSerXaaXaaXaaXaaProXaa                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= Xaa is pyroglutamyl or                          acetylsarcosine                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= Xaa is Trp or                                   3-(2- naphthyl)alaninyl                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= Xaa is N-methyl-Ala, Tyr,                       N-methyl- Tyr, Ser, N-e-2-propyl-lysinyl,                                     (4'-chlorophenyl)alaninyl, His, Asn, Met, Ala, Arg or Ile                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= Xaa is an amino acid having a                   side chain comprising a cationic moiety                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= Xaa is Leu or Trp                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= Xaa is N-e-2-propyl-lysinyl,                    Gln, Met or Arg                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= Xaa is Gly-NH2                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       XaaHisXaaSerXaaXaaXaaXaaProXaa                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= Xaa is pyroglutamyl or                          acetylsarcosine                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= Xaa is Trp or                                   3-(2- naphthyl)alaninyl                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= Xaa is N-methyl-Ala, Tyr,                       N-methyl- Tyr, Ser, N-e-2-propyl-lysinyl,                                     (4'-chlorophenyl)alaninyl, His, Asn, Met, Ala, Arg or Ile                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= Xaa is an amino acid having a                   side chain comprising a receptor-modifying moiety                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= Xaa is Leu or Trp                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= Xaa is N-e-2-propyl-lysinyl,                    Gln, Met or Arg                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= Xaa is Gly-NH2                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       XaaHisXaaSerXaaXaaXaaXaaProXaa                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= Xaa is pyroglutamyl or                          acetylsarcosine                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= Xaa is Trp or                                   3-(2- naphthyl)alaninyl                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= Xaa is N-methyl-Ala, Tyr,                       N-methyl- Tyr, Ser, N-e-2-propyl-lysinyl,                                     (4'-chlorophenyl)alaninyl, His, Asn, Met, Ala, Arg or Ile                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= Xaa is an amino acid having a                   side chain comprising an N-acyl hydrophilic moiety                            (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= Xaa is Leu or Trp                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= Xaa is N-e-2-propyl-lysinyl,                    Gln, Met or Arg                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= Xaa is Gly-NH2                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       XaaHisXaaSerXaaXaaXaaXaaProXaa                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= Xaa is pyroglutamyl or                          acetylsarcosine                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= Xaa is Trp or                                   3-(2- naphthyl)alaninyl                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= Xaa is N-methyl-Ala, Tyr,                       N-methyl- Tyr, Ser, N-e-2-propyl-lysinyl,                                     (4'-chlorophenyl)alaninyl, His, Asn, Met, Ala, Arg or Ile                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= Xaa is an amino acid having a                   side chain comprising a small polar moiety                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= Xaa is Leu or Trp                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= Xaa is N-e-2-propyl-lysinyl,                    Gln, Met or Arg                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= Xaa is Gly-NH2                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       XaaHisXaaSerXaaXaaXaaXaaProXaa                                                1510                                                                          __________________________________________________________________________

What is claimed is:
 1. A peptide compound comprising a structure:

    A-B-C-D-E-F-G-H-I-J

wherein A is pyro-Glu, Ac-D-Nal, Ac-D-Qal, Ac-Sar, or Ac-D-Pal; B is Hisor 4-Cl-D-Phe; C is Trp, D-Pal, D-Nal, L-Nal-D-Pal(N-O), or D-Trp; D isSer; E is N-Me-Ala, Tyr, N-Me-Tyr, Ser, Lys(iPr), 4-Cl-Phe, His, Asn,Met, Ala, Arg or Ile; F is D-Asn or D-Gln; G is Leu or Trp; H isLys(iPr), Gln, Met, or Arg; I is Pro; and J is Gly-NH₂ or D-Ala-NH₂ ;ora pharmaceutically acceptable salt thereof.
 2. A peptide compoundcomprising a structure:

    A-B-C-D-E-F-G-H-I-J

wherein A is pyro-Glu, Ac-D-Nal, Ac-D-Qal, Ac-Sar, or Ac-D-Pal; B is Hisor 4-Cl-D-Phe; C is Trp, D-Pal, D-Nal, L-Nal-D-Pal(N-O), or D-Trp; D isSer; E is N-Me-Ala, Tyr, N-Me-Tyr, Ser, Lys(iPr), 4-Cl-Phe, His, Asn,Met, Ala, Arg or Ile; F is D-Asn; G is Leu or Trp; H is Lys(iPr), Gln,Met, or Arg; I is Pro; and J is Gly-NH₂ or D-Ala-NH₂ ;or apharmaceutically acceptable salt thereof.
 3. A peptide compoundcomprising astructure:Ac-D-Nal-4-Cl-D-Phe-D-Pal-Ser-N-Me-Tyr-D-Asn-Leu-Lys(iPr)-Pro-D-Ala-NH.sub.2;or a pharmaceutically acceptable salt thereof.
 4. A peptide compoundcomprising astructure:Ac-D-Nal-4-Cl-D-Phe-D-Pal-Ser-Tyr-D-Asn-Leu-Lys(iPr)-Pro-D-Ala-NH₂;or a pharmaceutically acceptable salt thereof.
 5. A pharmaceuticalcomposition comprising the peptide compound of any one of claims 1, 2, 3or 4, and a pharmaceutically acceptable carrier.